The instructions necessary for the proper development and function of each cell are encoded in the DNA the cell carries. However, DNA is susceptible to damage from toxic agents found in the environment. DNA damage can cause mutation, changes in the cellular instructions, which can lead to the development of cancer in humans. Nucleotide excision repair (NER) is a process that identifies and removes DNA lesions to prevent mutation. NER has the remarkable ability to identify many types of unrelated lesions, although this property is poorly understood. XPC is the primary lesion detecting protein in global genome NER (GG-NER). XPC binds to both single-stranded and double-stranded DNA but displays a preference for damaged duplex DNA. Determining how XPC interacts with DNA is critical for understanding how exposure to carcinogens, such as ultraviolet light and chemical commonly found in cigarette smoke, lead to cancer. In this proposal I will investigate the biochemical and structural interactions between XPC and DNA to determine how DNA lesions are identified in GG-NER. The hypothesis of this proposal is that XPC will bind any DNA that deviates from the normal structure but binds DNA containing bulky chemical lesions tighter. Two specific aims are designed to test this hypothesis. Aim 1 will use fluorescence anisotropy to determine the affinity of the XPC for DNA substrates containing different types of lesions to test whether the chemical identity of the lesion affects the ability of XPC to identify DNA damage sites. Aim 2 will elucidate the molecular interactions underlying DNA damage recognition in NER by determining the structure of XPC-damaged DNA complex by X-ray crystallography. In order to achieve these goals, the DNA binding apparatus of human XPC (XPCDNA) has been isolated and characterized. The XPCDNA construct reconstitutes the DNA binding properties of the full-length protein and maintains the ability to discriminate between damaged and undamaged DNA. However, the XPCDNA protein can be purified from E. coli in sufficient quantity and purity for structural biology studies. PUBLIC HEALTH RELEVANCE: This study will provide valuable new insight into how DNA damage is recognized in NER. Determining how DNA damage is detected will greatly improve our understanding of how mutation induces cancer in humans. Ultimately understanding how DNA damage is recognized has the potential to lead to advances in both cancer prevention and therapy.